The Parkinson Disease-associated Leucine-rich Repeat Kinase 2 (LRRK2) Is a Dimer That Undergoes Intramolecular Autophosphorylation

Elisa Greggio,Ibardo Zambrano,Alice Kaganovich,Alexandra Beilina,Jean-Marc Taymans,Veronique Daniëls,Patrick Lewis,Shushant Jain,Jinhui Ding,Ali Syed,Kelly J Thomas,Veerle Baekelandt,Mark R Cookson

doi:10.1074/jbc.m708718200

Abstract

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial and apparently sporadic Parkinson disease. LRRK2 is a multidomain protein kinase with autophosphorylation activity. It has previously been shown that the kinase activity of LRRK2 is required for neuronal toxicity, suggesting that understanding the mechanism of kinase activation and regulation may be important for the development of specific kinase inhibitors for Parkinson disease treatment. Here, we show that LRRK2 predominantly exists as a dimer under native conditions, a state that appears to be stabilized by multiple domain-domain interactions. Furthermore, an intact C terminus, but not N terminus, is required for autophosphorylation activity. We identify two residues in the activation loop that contribute to the regulation of LRRK2 autophosphorylation. Finally, we demonstrate that LRRK2 undergoes intramolecular autophosphorylation. Together, these results provide insight into the mechanism and regulation of LRRK2 kinase activity.

Highlights

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial and apparently sporadic Parkinson disease
It has previously been shown that the kinase activity of LRRK2 is required for neuronal toxicity, suggesting that understanding the mechanism of kinase activation and regulation may be important for the development of specific kinase inhibitors for Parkinson disease treatment
We have examined the autophosphorylation activity of the intact, full-length LRRK2 protein expressed in mammalian cells

Summary

EXPERIMENTAL PROCEDURES

Constructs and Cell Culture—Full-length LRRK2 was cloned to generate N-terminal 2x-Myc or GFP and C-terminal V5-His tagged proteins as described previously [10, 21]. Yeast Two-hybrid Screening—Yeast two-hybrid strain AH109 harboring the HIS3, ADE2, and lacZ reporters were transformed with a portion of LRRK2 (amino acids 1270 –1435) fused to the Gal DNA-binding domain This construct was determined to be negative in self-activation of all selection markers by co-transformation with empty vectors. GST Pulldowns—HEK293FT cells were transfected with GFP-tagged full-length LRRK2 or Myc-tagged truncated LRRK2 and lysed in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ␤-glycerophosphate, 1 mM Na3VO4, 1 ␮g/ml leupeptin, protease inhibitor mixture (Roche Applied Science). After four washes (phosphate-buffered saline, 150 mM NaCl, 1% Triton X-100, and protease inhibitors), copurified proteins were analyzed by immunoblot with anti-GFP or anti-Myc antibody (Roche Applied Science). Differences were considered significant when through the central ROC domain and the N terminus, we have p Ͻ 0.05

RESULTS

We further investigated whether

Findings

DISCUSSION